The glucocorticoids and glucagon interact to regulate gluconeogenesis in liver. The role of the glucocorticoids has been termed "permissive" since these hormones by themselves produce only a slight stimulation of gluconeogenesis but are required for full expression of the glucagon effects. The objective of the proposed research is to investigate the mechanism by which the glucocorticoids maintain the normal responsiveness of hepatocytes to hormones which act through cyclic AMP. Adult rat liver parenchymal cells in primary culture will be used since this cell system lends itself to the study of hormone action in which the hormones can be added singly, sequentially or simultaneously. These cells exhibit many of the differentiated characteristics expected of an adult liver parenchymal cell for up to two weeks in culture and are maintained in a chemically defined serum-free medium which permits the study of hormones free from the effects of the many endogenous hormones found in serum. Hormonal modulation of glucose production and amino acid transport will be correlated with changes in the cellular phosphoprotein profile. The subcellular location of the phosphoproteins of interest will be determined. Phosphorylation reactions will be carried out in vitro on subcellular fractions in order to study the factors influencing the phosphorylation-dephosphorylation of proteins which may be synthesized in response to glucocorticoid treatment. The effect of chronic changes in the culture medium (physiological versus elevated levels of glucose or the addition of fatty acids) on the cellular phosphoprotein profile in combination with acute changes in the hormonal status of the culture will be studied also. This study should provide useful information for further understanding the contribution of the liver to glucose homeostasis.